The immune system uses many mechanisms for attacking pathogens; however, not all of these mechanisms are necessarily activated after immunization. Protective immunity induced by vaccination is dependent on the capacity of the vaccine to elicit the appropriate immune response to resist or eliminate the pathogen. Depending on the pathogen, this may require a cell-mediated and/or humoral immune response.
The current paradigm for the role of helper T cells in the immune response is that they can be separated into subsets on the basis of the cytokines they produce, and that the distinct cytokine profile observed in these cells determines their function. This T cell model includes two major subsets: TH-1 cells that produce IL-2 and interferon xcex3(IFN-xcex3) which augment both cellular and humoral immune responses, and TH-2 cells that produce IL-4, IL-5 and IL-10 which augment humoral immune responses (Mosmann et al., J. Immunol. 126:2348 (1986)). It is often desirable to enhance the immunogenic potency of an antigen in order to obtain a stronger immune response in the organism being immunized and co strengthen host resistance to the antigen-bearing agent. A substance that enhances the immunogenicity of an antigen with which it is administered is known as an adjuvant. For example, certain lymphokines have been shown to have adjuvant activity, thereby enhancing the immune response to an antigen (Nencioni et al., J. Immunol. 139:800-804 (1987); EP285441 to Howard et al.).
This invention pertains to vaccine compositions comprising a mixture of one or more pneumococcal or meningococcal antigens, the interleukin IL-12 and a mineral in suspension. The IL-12 can be either adsorbed onto the mineral suspension or simply mixed therewith. In a particular embodiment of the invention, the IL-12 is adsorbed onto a mineral suspension such as alum (e.g., aluminum hydroxide or aluminum phosphate). These vaccine compositions modulate the protective immune response to the antigen; that is, the vaccine composition is capable of quantitatively and qualitatively improving the vaccinated host""s antibody response, and quantitatively increasing cell-mediated immunity for a protective response to a pathogen. In a particular embodiment of the invention, the antigen is a pneumococcal or meningococcal antigen; the antigens are optionally conjugated to a carrier molecule, such as in a pneumococcal or meningococcal glycoconjugate.
Studies described herein show that IL-12 can modify the humoral response of mice immunized with pneumococcal and meningococcal glycoconjugate vaccines formulated with aluminum phosphate (AlPO4) The particular pneumococcal polysaccharide serotypes exemplified herein are serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F, (Pn1, Pn4, Pn5, Pn6B, Pn9V, Pn14, Pn18C, Pn19F, Pn23F), and the meningococcal polysaccharide is type C (Men C). These serotypes, however, are not to be construed to limit the scope of the invention, as other pneumococcal and meningococcal serotypes are also suitable for use herein. Moreover, it will be apparent to the skilled artisan that conjugation to a carrier molecule, such as the CRM197 protein exemplified herein, is optional, depending upon the immunogenicity of the selected pneumococcal or meningococcal antigen.
Doses of IL-12 ranging from about 8 ng to about 1,000 ng increased the IgG1, IgG2a, IgG2b and IgG3 response to alum-adsorbed Pn14 or Pn6B. In addition they increased the IgG2a response to Pn4 and Pn9V. Doses of IL-12 of about 5,000 ng markedly reduced the overall IgG titers to Pn14, and especially the IgG1 and IgG2b titers.
The invention also pertains to methods for preparing an immunogenic composition or a vaccine composition comprising a mixture of antigen and IL-12 with a mineral in suspension. In particular, the IL-12 is adsorbed onto the mineral suspension. The invention also pertains to methods for eliciting or increasing a vaccinee""s IFN-xcex3-producing T cells and complement-fixing IgG antibodies for a protective immune response, comprising administering to a mammalian, e.g., human or primate, host an effective amount of a vaccine composition comprising a mixture of antigen, IL-12 and a mineral in suspension in a physiologically acceptable solution. In particular, the IL-12 is adsorbed onto the mineral suspension.
Work described herein reveals the ability of IL-12 to increase the immune response to alum-based pneumococcal vaccines, particularly serotype 14 and serotype 6B pneumococcal glycoconjugate vaccines, and meningococcal vaccines, particularly type C, to increase the proportion of complement-fixing IgG2a and IgG2b antibodies. As described herein, PnPs-14-CRM197 vaccine comprises a serotype 14 pneumococcal polysaccharide conjugated to a non-toxic mutant of diphtheria toxoid (cross-reacting material) designated CRM197, and PnPs6B-CRM197 vaccine comprises a serotype 6B pneumococcal polysaccharide conjugated to CRM197. IL-12 was compared to MPL(copyright) (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Inc., Hamitton, Mont.), which in mice is a potent adjuvant for pneumococcal vaccines. In a separate experiment conducted in Balb/c mice, the effect of IL-12 on the cytokine profile of the CRM-specific T cells induced by the exemplary conjugate vaccines on alum was examined.
IL-12 is produced by a variety of antigen-presenting cells, principally macrophaqes and monocytes. It is a critical element in the induction of TH-1 cells from naive T cells. Production of IL-12 or the ability to respond to it has been shown to be critical in the development of protective TH-1-like responses, for example, during parasitic infections, most notably Leishmaniasis (Scott et al., U.S. Pat. No. 5,571,55). The effects of IL-12 are mediated by IFN-xcex3 produced by NK cells and T helper cells. Interleukin-12 (IL-12), originally called natural killer cell stimulatory factor, is a heterodimeric cytokine (Kobayashi et al., J. Exp. Med. 170:827 (1989)). The expression and isolation of IL-12 protein in recombinant host cells is described in International Patent Application WO 90/05147, published May 17, 1990.
The studies described herein reveal the utility of IL-12 as an adjuvant in a pneumococcal or meningococcal vaccine, and particularly a pneumococcal or meningococcal glycoconjugate vaccine. Accordingly, this invention pertains to vaccine compositions comprising a mixture of such an antigen, IL-12 and a mineral in suspension. In a particular embodiment of the invention, the IL-12 is adsorbed onto a mineral suspension such as alum (e.g., aluminum hydroxide or aluminum phosphate). These vaccine compositions modulate the protective immune response to the antigen; that is, the vaccine composition is capable of eliciting the vaccinated host""s complement-fixing antibodies for a protective response to the pathogen. In a particular embodiment of the invention, the antigen is a pneumococcal antigen, particularly a pneumococcal polysaccharide; the pneumococcal antigen is optionally conjugated to a carrier molecule, such as in a pneumococcal glycoconjugate. The particular pneumococcal polysaccharide serotypes exemplified herein are serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F; however, these serotypes are not to be construed to limit the scope of the invention, as other serotypes are also suitable for use herein.
In another embodiment of the invention, the antigen is a meningococcal antigen, particularly a meningococcal polysaccharide; the meningococcal antigen is optionally conjugated to a carrier molecule, such as in a meningococcal glycoconjugate. Type C Neisseria meningitidis is exemplified herein; however, this type is not to be construed to limit the scope of the invention, as other types are also suitable for use herein.
IL-12 can be obtained from several suitable sources. It can be produced by recombinant DNA methodology; for example, the gene encoding human IL-12 has been cloned and expressed in host systems, permitting the production of large quantities of pure human IL-12. Also useful in the present invention are biologically active subunits or fragments of IL-12. Commercial sources of recombinant human and murine IL-12 include Genetics Institute, Inc. (Cambridge, Mass.).
The antigen of this invention, e.g., a pneumococcal or meningococcal antigen or a pneumococcal or meningococcal glycoconjugate, can be used to elicit an immune response to an antigen in a mammalian host. For example, the antigen can be a serotype 14 or 6B pneumococcal polysaccharide or a portion thereof which retains the ability to stimulate an immune response. Additional suitable antigens include polysaccharides from other encapsulated bacteria and conjugates thereof, secreted toxins and outer membrane proteins.
The method comprises administering to the mammal, e.g., human or primate, an immunologically effective dose of a vaccine composition comprising a mixture of an antigen, such as a pneumococcal antigen or a pneumococcal conjugate, and an adjuvant amount of IL-12 adsorbed onto a mineral in suspension.
As used herein, an xe2x80x9cimmunologically effectivexe2x80x9d dose of the vaccine composition is a dose which is suitable to elicit an immune response. The particular dosage of IL-12 and the antigen will depend upon the age, weight and medical condition of the mammal to be treated, as well as on the method of administration. Suitable doses will be readily determined by the skilled artisan. The vaccine composition can be optionally administered in a pharmaceutically or physiologically acceptable vehicle, such as physiological saline or ethanol polyols such as glycerol or propylene glycol.
The vaccine composition may optionally comprise additional adjuvants such as vegetable oils or emulsions thereof, surface active substances, e.g., hexadecylamin, octadecyl amino acid esters, octadecylamine, lysolecithin, dimethyl-dioctadecylammonium bromide, N,N-dicoctadecyl-Nxe2x80x2-Nxe2x80x2bis (2-hydroxyethyl-propane diamine), methoxyhexadecylglycerol, and pluronic polyols; polyamines, e.g., pyran, dextransulfate, poly IC, carbopol; peptides, e.g., muramyl dipeptide, dimethylglycine, tuftsin; immune stimulating complexes; oil emulsions; liposaccharides such as MPL(copyright) and mineral gels. The antigens of this invention can also be incorporated into liposomes, cochleates, biodegradable polymers such as poly-lactide, poly-glycolide and poly-lactide-co-glycolides, or ISCOMS (immunostimulating complexes), and supplementary active ingredients may also be employed. The antigens of the present invention can also be administered in combination with bacterial toxins and their attenuated derivatives. The antigens of the present invention can also be administered in combination with other lymphokines, including, but not limited to, IL-2, IL-3, IL-15, IFN-xcex3 and GM-CSF.
The vaccines can be administered to a human or animal by a variety of routes, including but not limited to parenteral, intradermal, transdermal (such as by the use of slow release polymers), intramuscular, intraperitoneal, intravenous, subcutaneous, oral and intranasal routes of administration. The amount of antigen employed in such vaccines will vary depending upon the identity of the antigen. Adjustment and manipulation of established dosage ranges used with traditional carrier antigens for adaptation to the present vaccine is well within the ability of those skilled in the art. The vaccines of the present invention are intended for use in the treatment of both immature and adult warm-blooded animals, and, in particular, humans. Typically, the IL-12 and the antigen will be co-administered; however, in some instances the skilled artisan will appreciate that the IL-12 can be administered close in time cut prior to or after vaccination with the antigen.
The pneumococcal and meningococcal antigens of the present invention can be coupled to a carrier molecule in order to modulate or enhance the immune response. Suitable carrier proteins include bacterial toxins rendered safe by chemical or generic means for administration to mammals and immunologically effective as carriers. Examples include pertussis, diphtheria, and tetanus toxoids and non-toxic mutant proteins (cross-reacting materials (CRM)), such as the non-toxic variant of diphtheria toxoid, CRM197. Fragments of the native toxins or toxoids, which contain at least one T-cell epitope, are also useful as carriers for antigens, as are outer membrane protein complexes. Methods for preparing conjugates of pneumococcal antigens and carrier molecules are well known in the art and can be found, for example, in Dick and Burret, Contrib Microbiol Immunol. 10:48-114 (Cruse J M, Lewis R E Jr, eds; Basel, Krager (1989)) and U.S. Pat. No. 5,360,897 (Anderson et al.).
The adjuvant action of IL-12 has a number of important implications. The adjuvanticity of IL-12 can increase the concentration of protective functional antibodies produced against the antigen in the vaccinated organism. The use of IL-12 as an adjuvant can enhance the ability of antigens which are weakly antigenic or poorly immunogenic to elicit an immune response. It may also provide for safer vaccination when the antigen is toxic at the concentration normally required for effective immunization. By reducing the amount of antigen, the risk of toxic reaction is reduced.
Typically, vaccination regimens call for the administration of antigen over a period of weeks or months in order to stimulate a xe2x80x9cprotectivexe2x80x9d immune response. A protective immune response is an immune response sufficient to protect the immunized organism from productive infection by a particular pathogen or pathogens to which the vaccine is directed.
As shown in the Examples, in an alum-formulated vaccine, comprising IL-12 adsorbed onto AlPO4 and a serotype 14 or serotype 6B pneumococcal polysaccharide conjugated to CRM197, which normally induces a response dominated by IgG1, 0.2 xcexcg of IL-12 substantially increased the IgG2a and IgG3 subclasses in both Balb/c and Swiss Webster mice, but had little or no effect on IgG1. Enhancement of IgG2b to Pn14 was seen with Swiss Webster mice; 0.2 xcexcg of IL-12 had the same effect as 25 xcexcg of MPL(copyright) on the IgG subclass response to Pn14, suggesting that IL-12 is at least 100-fold more biologically active than MPL(copyright) in this regard. As expected from the IgG subclass distribution, especially the enhanced IgG2a response, the opsonophagocytic activity of the antisera for Pn14 pneumococci from mice receiving 0.2 xcexcg IL-12 was higher than that of controls and was equivalent to that of mice immunized with vaccine formulated with a much larger amount of MPL(copyright).
Briefly, IgG2a and IgG2b antibodies are very efficient at activating the complement system, whereas IgG1 antibodies are not. The complement system consists of a series of plasma proteins which come together around IgG2a or IgG2b bound to antigen (e.g., bacteria) to form a large molecular complex. Deposition of this complex on the surface of bacteria results in the killing of the bacteria by perforating the cell membrane (bactericidal activity) or by facilitating the recognition of the bacteria by phagocytic cells (such as polymorphonuclear cells (PMN) used in this study), which take up the bacteria and kill them (opsonophagocytosis).
Increasing the dose of IL-12 profoundly reduced the IgG1 and IgG2b responses. The reduction in these immunoglobulin subclasses was not simply due to a change in the kinetics of the antibody response, as has been observed in the hen egg lysozyme (HEL) system (Buchanan, Van Cleave and Metzger, Abstract #1945; 9th International Congress of Immunology (1995)), as these subclasses were reduced at all time points tested. The effect on IgG1 was expected given that switching of B cells to this subclass requires IL-4, a TH-2 cytokine whose production is inhibited by IL-12. The reduction in IgG2b, however, was not expected since in previous studies increased levels of IgG2b have correlated with the presence of TH-1-like T cells. It is likely that cytokines other than, or in addition to, IFN-xcex3 are involved in regulation of IgG2b. For example, Germann et al. (Eur J. Immunol 25:823-829 (1995)) found that treating mice with anti-IFN-xcex3 inhibited the ability of IL-12 to promote IgG2a responses, but not IgG2b. Other studies have implicated TGF-xcex2 as an important factor in the induction of IgG2b (reviewed by J. Stavnezer, J. Immunol. 55:1547-1651 (1995)). Without wishing to be bound by theory, it is possible that high doses of IL-12 may affect TGF-xcex2 production or responsiveness to it.
IFN-xcex3 is critical for the induction of IgG2a antibodies to T-dependent protein antigens (Finkelman and Holmes, Annu. Rev. Immunol. 8:303-33 (1990)) and IgG3 responses to T-independent antigens (Snapper et al., J. Exp. Med. 175:1367-1371 (1992)). Increased IFN-xcex3 response was consistently found after a single vaccination with vaccine (PnPs-14-CRM197) containing IL-12 and AlPO4 and after boosting. The effect of IL-12 on the TH-2 cytokines IL-5 and IL-10 appears to depend on when the lymphoid cells are harvested after vaccination, and possibly on the particular cytokine. Exogenous IL-12 completely abolished antigen-specific IL-5 and IL-10 production by lymph node cells (LNC) harvested 1 week after primary vaccination. After secondary vaccination, differences were seen between these two cytokines; IL-5 production by either LNC or splenocytes was completely abolished by 1 xcexcg IL-12 in the vaccine, but IL-10 production was largely unaffected after boosting. It is unclear whether these differences are due to setting up the cultures at different times or reflect the expansion of a TH-2-like population upon subsequent revaccination. The latter possibility is consistent with data from Wolf and colleagues (Bliss et al., J. Immunol 156:887-894 (1996)), indicating that IL-4-producing T cells can be recovered from Balb/c mice previously immunized with vaccine containing IL-12 and boosted with soluble antigen. In their studies, IL-4 was detected even if IL-12 was included in the secondary vaccine. The presence of TH-2 cytokines after boosting may explain why, in Balb/c mice, even high levels of IL-12 could not reduce the secondary IgG1 response to below control levels (conjugate vaccine on alum). Unlike the Balb/c mice, high doses of IL-12 severely inhibited the IgG1 response of Swiss Webster mice. Whether this is associated with decreased production of TH-2 cytokines after the second vaccination is unclear.
In the present studies, IL-12 exhibited either only immunomodulatory activity or behaved both as a xe2x80x9cclassicalxe2x80x9d adjuvant, and a immunomodulator, depending on the vaccine. In the study with PnPs14-CRM197 the IgG response (especially the primary response) to the vaccine was not substantially elevated by the presence of the cytokine but certain subclasses, i.e. IgG2a and IgG3, were elevated whereas the others were unchanged or diminished. Thus, IL-12 is useful for altering the humoral response to an already immunogenic vaccine. It is possible that in these studies the adjuvant activity of IL-12 was masked by the presence of alum, which is an adequate adjuvant on its own for the highly immunogenic PnPs-14 conjugate. The adjuvanticity of IL-12 may be better demonstrated in the absence of alum, by reducing the dose of conjugate or by using a poorly immunogenic conjugate. Thus, further evaluations were carried out using IL-12 in the presence and absence of alum with PnPs6B conjugate vaccines, which are less immunogenic in Swiss Webster mice than PnPs-14 conjugate vaccines.
An additional study was designed to address the issue of IL-12 adjuvant activity for a poorly immunogenic pneumococcal conjugate. The Pn18C conjugate was chosen, as it is poorly immunogenic when formulated with AlPO4, i.e., it induces low IgG Liters and not all mice respond to it. When formulated with MPL or QS-21, higher IgG fibers and a greater frequency of responders can be achieved.
One hundred xcexcg MPL(copyright) plus AlPO4 or 20 xcexcg QS-21(trademark) were the best adjuvants in this study for a Pn18C response as they induced the highest frequency of responders to this serotype. Nonetheless, IL-12 had marked effects on the IgG response to the carrier protein, CRM197, in mice immunized with this conjugate. Moreover, the effects of the cytokine were modified by the presence of AlPO4 in the vaccine. IL-12 clearly acted as an adjuvant for vaccines formulated without AlPO4, causing a dose-dependent increase in IgG titers after primary and secondary vaccination. IL-12 enhanced the IgG2a response to CRM197, which is consistent with its ability to favor the induction of TH-1-like helper cells (IFN-xcex3 producers). However, IL-12 also enhanced the IgG1 response to CRM197 after primary and secondary vaccination. IgG1 antibodies are normally associated with TH-2-like helper cells whit. produce IL-4.
Inclusion of 0.1 xcexcg IL-12 into an AlPO4-based Pn18C conjugate vaccine (which on its own induced a 10-fold higher CRM197 response) had no effect on IgG1 but substantially increased the IgG2a titer. The IgG2a titer achieved with 0.1 xcexcg IL-12 was at least as high as that obtained with 5 xcexcg IL-12 the absence of AlPO4. It should be noted, however, that the presence of AlPO4 does not preclude the enhancement of IgG1 responses by IL-12. In mice immunized with the Pn14 conjugate on AlPO4, a 0.2 xcexcg dose of IL-12 enhanced the IgG1, IgG2a and IgG2b titers to CRM197. The differences in the effect on IgG1 may reflect differences in the immunogenicity of the two conjugates for CRM197 IgG responses; the Pn14 conjugate on AlPO4 induced 10-fold lower CRM197 IgG titers so that there was room for IL-12 to enhance an IgG1 response, but not when mice were immunized with Pn18C conjugate on AlPO4. The fact that MPL(copyright) and QS-21(trademark) markedly increased the IgG1 titers in mice immunized with Pn18C conjugate on AlPO4 indicates that the IgG1 response had not been maximally stimulated. Alternatively, the nature of the saccharides on the conjugates may be a factor. In both experiments, higher doses of IL-12 resulted in a marked diminution of the IgG1, IgG2a and IgG2b tiers to CRM197, an effect that was not seen in the absence of AlPO4.
IL-12 probably exerts its adjuvant effect differently than MPL(copyright) or QS-21(trademark). IL-12 markedly enhanced the CRM197,9, IgG2a titers in mice immunized with Pn18C conjugate but had minimal effects on IgG2b. In contrast, MPL(copyright) and QS-21(trademark) enhanced the titers of both IgG subclasses. The dissociation of these two subclasses suggests that IgG2b is induced by cytokines other than, or in addition to, the IFN-xcex3 that drives switching to IgG2a and is known to mediate the immunomodulatory effects of IL-12. One candidate for driving IgG2b production is TGFb. The nature of the antigen cannot be excluded, however, since in mice immunized with Pn14 conjugate, 0.2 xcexcg IL-12 caused IgG2a and IgG2b to be elevated to similar levels which were equivalent to the titers promoted by 25 xcexcg MPL(copyright).
Studies utilizing a bivalent vaccine consisting of a PnPs14-CRM197 conjugate mixed with a conjugate of capsular polysaccharide from serotype 6B pneumococci covalently linked to CRM197 (PnPs6B-CRM197) confirmed and extended the above-described findings. IL-12 not only modified the IgG response to the Pn6B conjugate, but also enhanced the overall IgG titer to the conjugate. Moreover, this work further demonstrates that the adjuvant activity of relatively low doses of IL-12 is enhanced by formulating it with AlPO4. Unlike the above-described studies with PnPs-14-CRM197 glycoconjugate, IL-12/AlPO4 enhanced both the IgG1 and IgG2a subclasses to Pn6B, indicating that the apparent lack of enhancement of the Pn14 IgG1 response by IL-12 is probably not a generalizable phenomenon. This work further supports the idea that the mechanisms of adjuvant activity by IL-12 and MPL(copyright) are not equivalent. Both adjuvants enhanced the Pn6B IgG1 and IgG2a titers to similar levels, but MPL(copyright) was more effective at promoting IgG2b and IgG3 antibodies.
IL-12/AlPO4 did nor act as an adjuvant for the Pn14 IgG response. The reason for this is not clear; however, without wishing to be bound by theory, this most likely reflects the fact that in previous studies mice were immunized with a 1 xcexcg dose of PnPs-14-CRM197 glycoconjugate, i.e., 10-fold higher than in the Pn6B studies. The applicability of IL-12 to more complex pneumococcal vaccines was demonstrated using a nonavalent vaccine containing glycoconjugates from serotype 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F pneumococci. The combination of IL-12 with AlPO4 enhanced the IgG2a antibodies to PnPs4 and PnPs9V, in addition to PnPs6B and PnPs14, and increased the ability of mice to respond to glycoconjugate prepared with serotype 18C pneumococcal saccharide (PnPs-18C-CRM197) which is poorly immunogenic in mice.
In further examples, IL-12 was tested with a glycoconjugate vaccine against type C Neiserria meningitidis (MenC) and a glycoconjugate vaccine against type B Hemophilus influenzae (HbOC). Formulating that vaccine with 50 ng IL-12 and AlPO4 enhanced the IgG2a titers to MenC capsular polysaccharide although not to HbOC.
The data presented herein indicate that AlPO4 can greatly enhance the potency of IL-12 so that substantially lower doses of the cytokine can be used. One possible mechanism is that IL-12 binds to AlPO4, thereby enhancing its persistence in the animal; additional studies indicate that IL-12 rapidly binds to alum (data not shown). Alternatively, the local inflammatory effect of AlPO4 may induce cytokines that potentiate the biological activity of IL-12.
In addition to understanding the physical interaction of IL-12 with AlPO4, several other issues arise from the present work with pneumococcal vaccines formulated with IL-12. Given that AlPO4 enhances the activity of IL-12, it would be useful to know the minimal dose of cytokine needed to adjuvant the IgG response to pneumococcal glycoconjugates, as well as whether IL-5-producing T cells are activated by IL-12-containing glycoconjugate vaccines. These two questions were addressed in the studies in Balb/c mice described in Example 4.